Project Summary/Abstract Tumor-specific CD8 T cell peripheral tolerance can be a major barrier to the generation of potent anti- tumor immunity. Recent studies have begun to examine whether signaling through co-stimulatory molecules can sufficiently boost the immune response to reverse tumor-specific tolerance and promote anti-tumor immunity. To this end, our laboratory and others have focused on the mechanisms through which ligation of the OX40 (CD134) co-stimulatory molecule, a member of tumor necrosis factor receptor (TNFR) super-family, augments CD4 and CD8 T cell expansion, differentiation, and survival. Importantly, several studies have also shown that OX40 is expressed on T cells isolated from the tumor-draining lymph nodes of tumor-bearing hosts and that OX40 engagement can boost anti-tumor immunity in vivo. OX40-mediated signaling has also been shown to overcome peptide-induced CD4 T cell anergy. Recently, we demonstrated that OX40 ligation could restore the function of anergic tumor-reactive CD8 T cells in vivo. Although anti-OX40 therapy led to partial tumor regression, the tumors ultimately recurred. Thus, understanding the mechanisms regulating the induction of tumor-specific anergy may lead to the development of new therapeutic strategies to enhance CD8 T cell-mediated anti-tumor immunity. In Aim I of this proposal, we will investigate the mechanisms by which the common gamma chain (gc) cytokines IL-2 and IL-4 regulate OX40 receptor expression on CD8 T cells including the molecular mechanisms regulating activation of the OX40 promoter. In Aim II, we will determine the molecular mechanisms by which tumors induce CD8 T cell anergy and test the hypothesis that combined anti-OX40/gc cytokine therapy can restore the function of anergic CTL in tumor-bearing hosts. Aim III seeks to test the hypothesis that anti-OX40 therapy can enhance the differentiation of endogenous tumor-specific CD8 T cells in mice with spontaneously arising prostate cancer and to test whether anti-OX40 therapy promotes the differentiation of tumor-reactive CD8 T cells in cancer patients that are currently being treated with an agonist anti-OX40 mAb in a phase I clinical trial at the EACRI. Taken together, these studies will provide insight into the mechanisms regulating OX40 expression, the molecular basis of tumor- specific CD8 T cell anergy, and whether anti-OX40 therapy can augment the endogenous CD8 T cell response in both tumor-bearing mice and cancer patients.